1. Field of the Invention
The present invention relates to a highly sensitive and convenient method using a capillary column as a bioseparator/bioreactor for detection of microbial contamination in food products.
2. Brief Description of the Related Art
Microbial contamination in food products is a major concern to the food industry, regulatory agencies and consumers [S. L. Wilkinson, C and CE News. 75 (1997) 24]. Foodborne illness caused by pathogenic microorganisms poses a serious threat to public health. The Center for Disease Control and Prevention (CDC) estimated that annually 76 million illnesses, 325,000 hospitalized and 5,000 deaths were caused by bacterial contamination of food products in the United States. It is imperative to develop more effective and rapid technology to detect specific pathogens such as Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes to ensure that safe food products reach the public [M. W. Griffiths, J. AOAC Int. 80 (1997) 1143] [H. Van der Zee, et al., J. AOAC Int. 80 (1997) 934] [P. C. Vasavada, Food Testing and Analysis. 47 (1997) 18]. Escherichia coli O157:H7 is one of the most dangerous pathogens [R. L. Buchanan, et al., Food Technol. 51 (1997) 69] [P. M. Griffin, et al., Epidemiol. Rev. 13 (1991) 60], and it has been associated with two important human diseases: hemorrhagic colitis and hemolytic uremic syndrome (HUS). It can be transmitted via contaminated foods such as raw ground beef and unpasteurized milk [Center of Disease Control, Morbid. Mortal. Weekly Rep. 42 (1993) 85] and from person to person [Center of Disease Control, Morbid. Mortal. Weekly Rep. 42 (1993) 85], e.g., in nursing houses and day care facilities. Salmonella Typhimurium, one of the pathogens most likely to be found in commonly slaughtered livestock (cattle, sheep, and swing) and poultry (chicken and turkey), causes acute diarrhea, vomiting, abdominal pain, and fever. Occasionally, it may cause blood stream infection and death. Symptoms occur 6-72 hours after eating contaminated foods [USDA Food Safety and Inspection Service (FSIS). 1999, 26 Aug.]. Salmonella contamination in food products also results in the large economical burden for the industry due to products recalls.
Conventional microbiological culture methods used for bacterial detection are cumbersome and time-consuming, requiring 3-4 days for confirmation. Immunosorbent assays also suffer from complexities in sample pretreatment and measuring procedures, because they use isotopes and are susceptible to interference caused by chromogens in food samples. Even though some methods have been developed in an effort to replace traditional techniques, the enzyme-linked immunosorbent assays (ELISA), which can achieve high specificity and sensitivity, still needs 24 hours or more to achieve the necessary detection limits [K. S. Cudjoe, et al., J. Food Microbiol. 27 (1995) 11].
Some methods have been developed for the rapid detection of E. coli O157:H7 in an effort to replace conventional techniques [E. D. Boer, et al., J. Appl. Microbiol. 88(S) (2000) 133S]. Several procedures, such as antibody direct epifluorescent filter technique [L. Restaino, et al., J. Food Prot. 59 (1996) 1072] [L. Restaino, et al., Lett. Appl. Microbiol. 24 (1997) 401], ATP bioluminescence [M. W. Griffiths, J. Dairy. Sci. 76 (1993) 3118], enzyme-linked immunosorbent assays [R. P. Johnson, et al., Appl. Environ. Microbiol. 61 (1995) 386] [P. M. Fratamico, et al., J. Food Prot. 61 (1998) 934] [J-M. Woody, et al., J. Food Prot. 61 (1998) 110] [N. V. Padhye, et al., Appl. Environ. Microbiol. 57 (1991) 2693], impedance [J. Dupont, et al., J. Appl. Bacteriol. 80 (1996) 81] [K. O. Colquhoun, et al., J. Appl. Bacteriol. 79 (1995) 635], multiplex polymerase chain reaction [J. P. Mckillip, et al., J. Appl. Microbiol. 89 (2000) 49] [P. M. Fratafico, et al., J. Clin. Microbiol. 33 (1995) 2188] [C. M. Gooding, et al., J. Dairy Research 64 (1997) 87] [J. L. Mckillip, et al., J. Food Prot. 63 (2000) 855], and flow cytometry [K. H. Seo, et al., J. Food Prot. 61 (1998) 812], have been reported. These procedures could reduce analysis time and give presumptive results within several hours to one day, when compared with cultural plating procedures that require two or more days. The detection limit of these methods varied from 103 to 105 cells/ml. Since E. coli O157:H7 cells in foods are usually present in small numbers, pre-enrichment is necessary to obtain a detectable signal for target bacteria in applications of these methods.
Kim and Park [I. S. Park, et al., Biosens. Bioelectron. 13 (1998) 1091] developed piezoelectric biosensors for detection of Salmonella in the range of 9.9×105 to 1.8×108 CFU/ml. Seo et al. [K. H. Seo, et al., J. Food Prot. 62 (1999) 431] developed an integrated optic interferometer system for detection of Salmonella in the range of 1.0×105 to 1.0×107 CFU/ml by observing the fringe shift generated by refractive index variation. By using membrane separation and electrochemical analysis, a biosensor possessed a linear response for S. Typhimurium from 5.0×103 to 5.0×106 CFU/ml within 2 hours [Z. P. Yang, et al., Electroanalysis 10 (1998) 913]. When immunomagnetic separation and electrochemical detection were applied, a biosensor system could be able to detect S. Typhimurium from 1.0×103 to 1.0×107 [Y. H. Che, et al., J. Rapid Meth. Auto. Microbiol. 7 (1999) 47] [Y. H. Che, et al., J. Food Prot. 63 (2000) 1043]. Brewster et al. [J. D. Brewster, et al., Anal. Chem. 68 (1996) 4153] prepared immunoelectrochemical sensors for the detection of Salmonella. Chang et al. [Y. H. Chang, et al., Biosci. Biotechnol. Biochem. 60 (1996) 1571] constructed a compact fiber optic-based biosensor for detection of Salmonella aureus by measuring laser light signal at 488 nm. These methods make it possible to miniaturize immunosensors in detection of bacteria. However, each of these methods has its particular disadvantages such as high detection limit and poor specificity, high cost of instruments and materials, and/or time-consuming.
Capillary columns offer the advantage of better surface-to-volume interaction and the reduced amount of reagent. More importantly, it would take shorter time for molecules to reach the surface in a capillary, resulting in a faster assay. Capillary columns have proven to be very successful in separation techniques [C. F. Poole, et al., Chromatography Today, Elsevier Science: New York, N.Y. 1991], but there are few reports on their applications in the separation and detection of pathogens. Recently, capillary columns were used as a bioseparator/bioreactor by chemically immobilizing anti-E. coli O157:H7 antibodies onto the inner wall of the column [Y. Liu, et al., Anal. Chem. 73 (2001) 5180]. After a sample and alkaline-phosphatase-labeled antibodies passed through the column and the “sandwich” immunocomplexes were formed, a substrate, p-phenol phosphate was added and then the absorbance of the enzymatic products was measured. A detection limit of 500 CFU/ml was obtained.
The invention described herein comprises a flow-injection analysis system with a bienzyme biosensor used to detect the product of enzymatic reaction instead of optical measurement. Phenol produced from the enzymatic reaction between alkaline phosphatase and its substrate, phenol phosphate was detected by a tyrosinase-horseradish peroxidase biosensor. Compared to UV spectroscopy, electrochemistry provided more sensitive detection [R. Q. Thompon, et al., Anal. Biochem. 192 (1991) 90]. With optimized conditions, a detection range from 8.8×101 to 8.8×106 CFU/ml was obtained for E. coli O157:H7, and the assay time was less than 2.5 hours without any enrichment. Capillary column has been used as a bioseparator/bioreactor for the detection of E. coli O157:H7 and a detection limit of 8.8×101 CFU/ml was obtained [Z. Y. Zhang, et al., Anal. Chim. Acta, 2002 (submitted)] [Y. Liu, et al., Anal. Chem. 73 (2001) 5180]. The capillary immunosensor was also used to detect S. Typhimurium with a total assay time less than 2 hours without any enrichment and a detection limit of 102 cfu/ml.
We demonstrate that this method is more sensitive and convenient compared to other methods developed for the detection of microbial contamination because capillary columns can offer the advantage of high surface-to-volume interaction.
References mentioned in this background section are not admitted to be prior art with respect to the present invention.